The Crystal Structure of the Family 6 Carbohydrate Binding Module from Cellvibrio mixtus Endoglucanase 5A in Complex with Oligosaccharides Reveals Two Distinct Binding Sites with Different Ligand Specificities

Abstract

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain noncatalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis.

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…This 3-fold helix was confirmed in the X-ray structure of a family 15 CBM, which employed a twisted platform [67]. The same oligosaccharide conformation was observed in two family 6 CBMs, which have a twisted sandwich conformation of aromatic amino acid side chains in their binding sites [61,63]. Somewhat surprisingly, the conformations of cello-oligosaccharides in the X-ray crystal structures of a family 4 (sandwich platform) [68], a family 17 (twisted platform) [60], and a family 29 CBM (twisted platform) [69] revealed a consistent turn in the chain, but neither a 2-nor 3-fold axis, in contrast to the perfect 2-fold helix observed in the chains of crystalline cellulose.…”

“…For example, the Type B CBM6 module of the Clostridium stercorarium xylanase has a very similar fold to the Type C lectin-like CBM32 family [61], but apparently binds longer oligosaccharide ligands. Furthermore, the Cellvibrio mixtus Cel5A CBM6 contains two discrete binding sites that display characteristics of Type B and Type C modules respectively [62,63]. Nevertheless, it is apparent that the hydrogen-bonding network between protein and ligand is more extensive in Type C CBMs than Type B modules, consistent with their lectin-like properties (see below).…”

The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

“…The mutation of Tyr 152 to alanine had no apparent effect on binding to the insoluble ligands. Taken together, these results suggest that the groove located in CtCBM11 is the carbohydrate-binding cleft and that residues Tyr ecule, whereas the fourth subsite displays exclusive specificity for a ␤-1,3-linked glucose (35). Analysis of the CtCBM11 structure, together with the mutagenesis data, indicates that unlike CBM6, the family 11 module contains only a single ligandbinding site, in common with the majority of CBMs characterized to date.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…A more extreme example of how topological changes can cause a dramatic change in ligand specificity is evident in site 1 in CBM6 modules. This site may adopt a pocket-like topology and thus recognize the termini of polysaccharide chains (Pires et al, 2004) or display an open cleft and bind to the internal regions of xylan (Czjzek et al, 2001). From the discussion above, it is apparent that CBMs, in common with lectins, display preformed carbohydrate-recognition sites that mirror the solution conformations of their target ligands, thereby minimizing the energetic penalty paid upon binding.…”

“…In some of the 59 CBM families, exemplified by CBM1, CBM10, and CBM20, ligand specificity is invariant (Linder and Teeri, 1997;Southall et al, 1999;Raghothama et al, 2000), while in some families, such as CBM6 (Czjzek et al, 2001;Pires et al, 2004), CBM4 (Boraston et al, 2002b), and CBM35 (Tunnicliffe et al, 2005;Montanier et al, 2009b), carbohydrate recognition is highly variable. In addition to defining a phylogenetic relationship between CBMs by clustering these modules into sequence-based families, they have also been classified into three categories (types A, B, and C) based on the topology of their ligand-binding sites and their mode of ligand recognition (for review, see Boraston et al, 2004; Fig.…”